Recovery of protein from



Patented Dec. 26, 1950 F F I CE RECOVERY OF PROTEIN FROM KERATINOUSMATERIALS Ernest C. Koerner, Wood Ridge, N. J.

No Drawing. Application February 26, 1949, Serial No. 78,663

11 Claims.

This invention relates to new anduseful improvements in the recovery ofprotein from keratincus materials.

Keratinous materials such as hair, hoofs, horns, feathers, wool and thelike, may be subjected to a cystine reducing treatment to thereby obtaina desirable protein. The cystine reduction involves the conversion ofthe cystine linkage to cysteine. Sodium sulfide (NazS) at an alkaline pHhas been successfully used in the past for the cystine reducingtreatment. Normally, keratinous material, however, will, as such, notreadily disperse in the sodium sulfide treating medium so that thedesirable intimate contact between the reactants is not obtainedresulting in an incomplete reaction and low yields. For the pmpose ofimprovin the disnersibility of the keratinous materials, their acidpre-treatment has been proposed. Such pre-treatment may, for instance,comprise the use of concentrated sulfuric acid or of formic orhydrochloric acid.

Both the hitherto propo ed acid pre-treatment of keratinous materialsand the sodium sulfide cystine reduction are subiect to certaindisadvantages and drawbacks. If, in the ac d pretreatment, aconcentrated acid is used, relatively stablesalts of the acids with akeratin are formed and a partial deterioration of the peptide linkagesoccurs resulting in an appreciable loss of material available forcystine reduction. Furthermore, the protein salts have differentsolubility properties and indicate per so an alteration of the protein.On the other hand if relatively dilute acids, such as formic acid andhydrochloric acid, are used, although no appreciable salt formation maytake place under certain conditions and although they tend to "softendiflicultly soluble parts of the keratinous material as, for instance,the epidermal layer and cortical fibrils of wool, they do exercise aconsiderable solvent act on on the keratinous material resulting in aloss thereof and thus of ultimate yield of protein upon cystinereduction.

The sodium sulfide cystine reducing treatment requires relatively largeamounts of this chemical and an excess thereof is normally neccssary'toeffect a satisfactory reduction of the cystine grouping to cysteine. Onthe other hand, sodium sulfide possesses an appreciable hydrolytic andotherwise destructive influence upon protein so that the required excessof this material is reiiected in impaired yields of protein resultingfrom the cystine reduction.

One object of the instant inventioncomprises an improved method for therecovery ofprotein from keratinous materials.

Another object of the invention comprises an improved acid pre-treatmentof keratinous materials as part of the processing thereof for therecovery of protein. l

Still another object of the invention is an improved cystine reductionof acid pre-treated keratinous materials as part of the processingthereof for the recovery of protein.

The foregoing and still further objects of the invention will beapparent from the following description.

I have discovered that, when treating a keratinous material such ashair, hoofs, horns, feathers, wool, etc, with a polyvalent acid beingone of a 12-14 N phosphoric acid and 8-9 N sulfuric acid at roomtemperature, for a period of time and using an amount of acid sufficientto render the keratinous material readily dispersible in an alkalinesodium sulfide solution, only a negligible loss of keratinous materialoccurs. Whereas, for instance, the use of a 4-7 N hydrochloric acidgives rise to a loss of approximately 15% by weight of keratinousmaterial (degreased wool) the treatment with either a 12 to 14 Nphosphoric acid or 8 to- 9 N sulfuric acid shows a loss of usu-- allynot more than about 2-3% by weight of the same material treated.Furthermore, analysis of the keratinous material treated with either thephosphoric acid or the sulfuric acid of the strength indicated showedthe complete absence of salt formation between the acid and the kerat n.

When app ying the acid pre-treatment in accordance with the invention,the range of normality of the acid medium is critical. The normality ofthe treating medium should be checked after the addition of the acid tothe keratinous material. In many cases by reason of alkaline residues orimpurities the normality of the medium may become reduced from that ofthe acid added. In such cases, the normality should be readjusted and bebrought back to within the range of 12-14 N for phosphoric acid or 8-9 Nfor sulfuric acid. The amount of acid should be suflicient to completelysubmerge the keratinous material in the same. An excess of acid isnormally not harmful. For most purposes, and particularly for W001, aratio by weight of 8 parts of the acid to one part Of the keratinousmaterial will suflice for proper submergence. The temperature should beroom temperature, i. e., a temperature within a broad range of fromabout iii-25 C. The completion of the acid pre-treatment is bestdetermined for any given keratinous substance by removing samples fromtime to time and checking these samples for ready dispersibility in asodium sulfide cystine reducing medium. In the majority of cases,however, a period of 60 hours will suffice for the completion of theacid pre-treatment, i. e., the conversion of the keratinous materialinto a form readily dispersible in a cystine reducing agent containingsodium sulfide.

In-the practice of the cystine reducing treat- 3 merit in accordancewith the invention a suitably acid pre-treated keratinous material, andpreferably a keratinous material subjected to acid pre-treatment withone of 12 to 14 N phosphoric acid and 8 to 9 N sulfuric acid, issubjected to the action of a mixture comprising 3 to 5% by weight of drykeratinous material sodium sulfide (NazS) and 3 to 6% by Weight of drykeratinous material sodium sulfoxylate (NazSzOr) at an alkaline pH ofabout 9 to 10 and at a temperature of less than 60 C. and preferably offrom -60 C. Heating is continued until the keratinous material issubstantially completely dissolved, which is, in most cases,accomplished after a period of heating of from 45-60 minutes. The hotsolution is then filtered and/or centrifuged and permitted to cool.Thereupon, the cold protein solution is precipitated with acid whileconstantly stirred. Best results are obtained by adding the acid throughthe hollow stem of a stirrer. The addition of acid is continued untilthe solution becomes substantiallyarigid mass which normally occurs whenthe pH reaches about a Value of 5 to 5.2. The end point of acidificationmay be visually determined by the appearance of curdling in the proteinmass, Within the preferred practice of the treatment in accordance withthe invention, I find it of advantage to add the sodium sulfide-sodiumsulfoxylate solution to the washed, acid pre-treated keratinousmaterial, stirring the cold mass until gelatinous and thereafter warmwith constant stirring to the desired reaction temperature. In the eventthat the sodium sulfide is used in the form of the usual commercialhydrate (QHzO) this should be taken into account and calculated withinthe above set forth percentage range for sodium sulfide as water free.

When the proceeding with the cystine reducing treatment in accordancewith the invention using the combination of sodium sulfide and sodiumsulfoxylate as hereinabove set forth, appreciably lesser amounts ofsodium sulfide are required than would be the case if sodium sulfidealone or in conjunction with some chemical other than sodium sulfoxylatewere used. The reaction mechanism involved in the cystine reduction whenproceeding in accordance with the instant invention may be exemplifiedas follows. The sodium sulfide (NazS) in the mixture reduce the cystinein wool to cysteine, liberating sulfur: I

NH NH The liberated sulfur is immediately taken up by the remaining NazSto form the disulfide:

Sodium sulfoxylate reacts spontaneously with the disulfide:

Thus, the sulfur liberated in the cystine to cysteine reduction icontinuously used for the regeneration of sodium sulfide (NazS) which isagainready to reduce the cystine to cysteine. In

this manner, appreciably lesser amounts of sodium sulfide can be used toeffectuate the same result and at the same time avoid all thedisadvantages attending the use of sodium sulfide in the normally largequantities. Furthermore, in addition to materially reducin thedeteriorating and hydrolytic influence of N azS on protein, savings perse in amount of material used are accomplished. Still further, oxidationby oxygen either originally in the dispersing bath or enter ing the openbath during dispersion, is also beneficially influenced by thesulfoxylate:

Towards the end of the cystine reduction, most of the sulfoxylate hasbeen used up and there is a tendency for the liberation of free sulfurin the solution, The presence, however, of the sodium sulfite (NazSOs)(see 3 and i above) produced in the regeneration of the sodium sulfide(see 3 above) functions to absorb any free sulfur readily formingtherewith sodium hyposulfite (NazSzOa) I N azSOs+S- NazS20a (5) Thus,when proceeding in accordance with the instant invention, the proteinprecipitated from the solution with acid (at a pH of about 5-5.2) issubstantially free of salts and sulfur and is recovered in exceptionallyhigh yields. All of the reaction products that finally result from thecystine reduction treatment in accordance with the invention aresubstantially water soluble and can be easilyremoved from the acidprecipitated protein by washing with water or other aqueous media at asubstantially neutral pH at which keratinous proteins are substantiallyinsoluble.

Analysis further showed that there was no evidence of. any deteriorationor modification of any of the amino acids that are normally aconstituent. part of keratinous substances.

The following examples are furnished by Way of illustration but not oflimitation.

Example I Take 900 cc. of phosphoric, acid having a strength of 13.0 N.The temperature, of this solution should be less. than 25 C.

Into this solution immerse, 1l0-120 grams of wool (containingapproximately 10% moisture); assure good wetting of the material andmixing of the. cid by evacuation or by some kind of stirring motion,Take care that all wool is covered by the liquid. Check again normalitywhich should not be below 12.0 N. If necessary,

' the normality should be readjusted and preferably to its originalstrength of 13.0 N. Finally close container and let stand. A sampledrawn after 60 hours and thoroughly washed with water was readilydispersible in an aqueous solution containing about 3.4% Na2SzO4, 9.4%NazSBHzO of a 10% solution; the pH of neutralization bath should noteven temporarily be greater than 8.

Let stand for at least one hour. then rinse thor oughly with cold water;the material may then be hydro-extracted and dried if desired.

Example 11 Take 200 cc. of concentrated sulfuric acid and make up byproper procedure to a total volume of 900 cc., adjust strength of acidto 8 N, temperature should be less than 25 C.

Into this solution immerse 110-120 grams of Wool (containingapproximately moisture) assure good wetting of the material and mixingof the acid by evacuation or by some kind of stirring motion. Take carethat all wool is covered by the liquid. Check again normality whichshould not be much below 8 N. If necessary, the normality should bereadjusted. Finally let stand. A sample drawn after 60 hours andthoroughly washed with water was readily dispersible in an aqueoussolution containing about 3.4% Nazszoi, 9.4% Na2S.9H2O and 2.4% NaOH.

Now drain acid as completely as possible, add approximately 200 cc. ofcold water (preferably by means of a spray) and drain again; bothdrainings should total approximately 900 cc. of

liquid. After readjustment to 8 N strength, this acid may be reusedagain for acid pretreatment. The wool is then washed with cold waterremoving thus most of the acid left in the material. For completeneutralization add slowly preferably in small portions 1-2 grams of sodaash in form of a 10% solution; the pH of neutralization bath should noteven temporarily be greater than 8. Let stand for at least one hour,then rinse thoroughly with cold water; the material may then behydro-extracted and dried if desired.

Example III 80 grams of acid pre-treated and neutralized wool(containing approximately 10% moisture) and obtained in the manner setforth in either Example I or II, are placed in a 2000 cc. beakerwhereupon 900 cc. of cold water is added and the material wetted out aswell as possible by suitable stirring. To this is then added 100 cc. ofan aqueous solution containing about 2.4 grams NaOH, 3.4 grams Na2S2O4(4.7% per weight of dry wool) and 9.3 grams Na2S.9H2O (4.2% NazS per dryweight of wool). The cold mass is stirred until gelatinous and isthereupon warmed with constant stirring to a temperature of about 50 C.(the temperature should be guarded against exceeding 60 C.). The pH ofthe solution should be approximately between 9 and 10 and preferably atabout 9.5. Temperature and stirring are maintained until the wool isdissolved. Substantial dissolution will be effected after about 45-60minutes. Thereafter, the solution is filtered and/or centrifuged whilehot.

After cooling, the cold centrifuged and/or filtered protein solution isnow precipitated in the conventional manner with, for instance, sulfuricacid. In order to achieve best results, it is preferred to add the acidthrough the hollow stem of the stirrer. Acid addition is continued untilthe solution becomes a substantially rigid mass which is normallyachieved at a pH of about 5.1. The end point of acid addition may beeasily observed by the appearance of curdling in the protein mass. It isrecommended that once this point is attained, a few ccs. of extra acidsolution be added under stirring to assure complete and uniformprecipitation.

The further working up of the precipitated protein is also carried on inthe conventional manner by separating the liquid from the protein,

draining, centrifuging or filtering the liquid, and washing theprecipitated protein' with minimum amounts of cold water having a pH ofapproximately 5.1 until a sample of cold wash water gives only a slightdiscoloration with silver nitrate solution (as test for hyposulfiteion). The protein is now ready for drying either in the air, in an ovenor in vacuo. The preferred method is drying in vacuo over caustic as itwill remove from the protein any trace of possibly unpleasant odor.

Example 1v Chicken feathers cleaned and defatted and acid pre-treated inaccordance with the procedure set forth in Examples I or II are used. 86grams of these acid pre-treated chicken feathers and containing aboutgrams of dry material are placed in 900 cc. of cold water and wetted outas well as possible by suitable stirring. To this is then added cc. ofan aqeuous solution containing 9.3 grams of NazS.9H2O (3.8% NazS per dryweight of keratin), 3.4 grams Na2S2O4 (4.2% per dry weight of keratin)and 2.5 grams NaOH (3.1% per dry weight of keratin) The cold mass isthen stirred until gelatinous and thereafter heated with constantstirring to a temperature of about 50 C. avoiding at any time that thetemperature exceeds 60 C., the pH substantially maintained around 9.5and heating and stirring continued until the feathers are dissolvedwhich takes place in about 45 to 60 min utes. The solution is thenfiltered or centrifuged while hot and the protein precipitated, washedand recovered as set forth in the preceding example.

Example V Cleaned and defatted filings from cow horn, and acidpro-treated in accordance with Exampics I or II, are used. 88 grams ofsuch acid pretreated filings and containing about 10% moisture areplaced in 900 cc. of cold water and wetted out as well as possible bysuitable stirring. There is then added 100 cc. of an aqueous solutioncontaining approximately 11.4 grams Na-2S.9H2O (4.6% Nazs per dry weightof keratin), 4.2 grams Na2SzO4 (5.2% Na2S2O4 per dry weight of keratin),and 3 grams NaOH (3.7% NaOH per dry weight of keratin). The cold mass ofthe horn material is then stirred until gelatinous and thereafter heatedwith constant stirring to a temperature of about 50 C. taking care thatthe temperature does not at any time exceed substantially 60 C. The pHis substantially maintained around 9.5. The heating and stirring arecontinued until the material is dissolved, which will take place inabout 45 to 60 minutes. The solution is then filtered or centrifugedwhile hot, and the protein precipitated is further Worked up for therecovery of the protein as set forth in Example III.

Within the preferred embodiment of my invention, best results areobtained particularly with respect to yield, quality and purity whenusing the combination of my novel acid pre-treatment in conjunction withthe herein set forth sodium sulfide-sodium sulfoxylate cystinereduction. It is to be understood, however, that my novel cystinereduction treatment may be used with the realization of the thereininherent sodium sulfide regeneration independent of the particularpretreatment to which a given keratinous material may have beensubjected to adapt the same to the NazS-NmSzO; cystine reduction. Thus,for

instance, a suitable keratinous material may be subjected to acidpre-treatment with, for exam pie, 2. 4 to '7 N HC1 solution. Though suchhydrochloric acid treatment involves the loss of approximately 15% ofkeratin, as against a loss of not more than about 2% of keratin whenutilizing the acid pro-treatment in accordance with my invention, theproduct resulting from the H01 treatment is dispersible in thesulfide-sulfoxylate solution in accordance with the invention and may besubjected to such treatment in accordance with any one of the aboveExamples III to V. In such cases, although the ultimate yield ofundeteriorated protein (calculated on the keratinous material used inthe acid pro-treatment) is'appreciably less than when proceeding inaccordance with the sulfuric acid or phosphoric acid treatment hereinspecified, the regeneration of sodium sulfide with its attendantadvantages permits the highly beneficial results of the treatment toassert themselves even with such HC1 pre-treated material.

As will be seen from the foregoing, my invention, within the broad scopethereof, essentially embraces in the recovery of protein from keratinousmaterials, the improvement which comprises subjecting a keratinousmaterial, pre treate for Nags cystine reduction, to the action of anaqueous solution, containing per dry weight of keratinous materialpresent about 3 to 5% News and about 3 to 6% Nag-S204, at a pI-I ofabout 9 to 10 and preferably about 9.5 and at a temperature of less than60 C. and preferably of from 20 to 60 C., the amount of aqueous mediumbeing sufiicient to substantially cover the keratinous material present,and the combined Na2S-Na2SzO4 concentration in said medium beingsufiicient for cystine reduction, preferably from 0.2-l% and for bestresults from about 05-03%, maintaining the reaction mass at said pH andat said temperature until the keratinous material is substantiallydissolved and recovering protein from said solution.

Within the broad preferred embodiment of the invention, the pro-treatedkeratinous material is an acid pre-treated elatinous material andpreferably a keratinous material pre-treated for Nags cystine reductionwith one of about 12 N-l4 N H3PO4 and about 8 N-Q N raise-4 andpreferably one of about 13 N HBPO-l and about 8 N H2SO4 at roomtemperature, and preferably between l825 C., the amount of acid usedbeing sufficient to substantially submerge the keratinous materialtherein, and maintaining the reaction mass at said acid normality untilthe heratinous material is substantially readiiv dispersible in anaqueous solution containing by weight thereof 25-35% NazS and .3. l%NazSzOa The remarkable results obtained in the cystine reduction inaccordance with the invention using the sodium sulfide-sulfoxylatecombination are particularly unexpected in view of the fact thatnormally mixtures of sodium sulfoxylat'e and sodium sulfide outside ofthe range of percentage specified therefor in accordance with theinstant invention possess a synergistic damaging efiect upon keratinousmaterials and particularly wool fibers which is considerably in excessof the individual damaging eilect exercised upon such materials byeither one of these chemicals alone even in their higher concentrations.It is within the very specific ranges and percentages here-- in recitedthat this damaging effect is substantially eliminated, thus giving riseto the highest possible yields of the reducing treatment of cystine tocysteine. Although for the sake of convenience and simplicity, NaOH hasbeen specified in the above formulae as participating in the reactioninvolving the regeneration of Na s, it is understood that the pI-Iof thereaction is the controlling factor in that at the alkaline pH of about 9to 10 specified for the cystine reducing treatment in accordance withthe invention, dissociated cations and OH ions are present in thesolution and they react as such irrespective of the specific nature ofthe cation present. Where reference, therefore, is made herein to the pHrange of 9 to 10 in connection with the cystine reducing treatment inaccordance with the invention, it is understood that it involves thepresence of the requisite amount of dissociated cations and OH ionscontrolling the particular pH and participating in the reactionmechanism as hereinabove exemplified.

I claim:

1. In the recovery of protein from keratinous materials the improvementwhich comprises subjecting a keratinous material, acid pro-treated forNazS cystine reduction, to the action of an aqueous solution, containingper dry weight of keratinous material present about 35% NazS and about3-6% NazSzOr, at a pH of about 9 to 10 and at a temperature of less than60 0., the amount of aqueous medium being sufiicient to substantiallycover the keratinous material present and the combined NazSNazS2O4concentration in said medium being sufiicient for cystine reduction ofsaid keratinous material, maintain ing the reaction mass at said pH andat said temperature until the keratinous material is sub stantiallydissolved and recovering protein from said solution.

2. Improvement in accordance with claim 1 in which said keratinousmaterial is subjected to the action of said aqueous solution at atemperature substantially from 20-60 C.

3. Improvement in accordance with claim 2 in which said combinedNa2SNazSzO4 concentration in said medium is substantially from 0.2 to1%.

4. Improvement in accordance with claim 3 in which said keratinousmaterial is subjected to the action of said aqueous solution at a pH ofabout 9.5 and in which said combined concentration in said medium isfrom about 0.5 to 0.8%.

5. In the recovery of protein from keratinous materials the improvementwhich comprises subj'ecting such material to the action of one member ofthe group consstin of about 12 N-l4 N H3PO4 and about 8 N-9 N H2804, theamount of acid used being sufiicient to substantially submerge thekeratinous material therein, and main taining the reaction mass at saidacid normality until the keratinous material is substantially readilydispersible in an aqueous solution containing by Weight thereof 25-35%NazS and .3-.4% Na2S2O4.

6. Improvement in accordance with claim 5 in which said keratinousmaterial is subjected to said acid treatment at a temperature from about18-25 C. and in which the reaction mass is maintained substantially atsaid temperature.

7. Improvement in accordance with claim 6 in which said keratinousmaterial is subjected to the action of one member of the groupconsisting of about 13 N H3PO4 and about 8 N H2304.

8. In the recovery protein from keratinous mew.

terials the improvement which comprises subjecting keratinous materialto the action of one member of the group consisting of about 12 N-14 NHaPO4 and about 8 N-9 N H2804 the amount of acid used being suflicientto substantially submerge the keratinous material therein, maintainingthe reaction mass at said acid normality until the keratinous materialis substantially readily dispersible in an aqueous solution containingby weight thereof 25-35% NazS and .3-.4% Na2SzO4, thereafter subjectingthe neutralized and washed thusly pre-treated keratinous material to theaction of an aqueous solution, containing per dry weight of keratinousmaterial present about 3-5% NazS and about 3-6% NazSzO; at a pH of about9 to 10 and at a temperature of less than 60 C., the amount of said lastmentioned aqueous medium being sufficient to substantially cover thekeratinous material present and the combined Na2SNa2S2O4 concentrationin said medium being sufiicient for cystine reduction, maintaining thereaction mass at said pH and at said temperature until the keratinousmaterial is substantially dissolved, and recovering protein from saidsolution.

9. Improvement in accordance with claim 8 in which said keratinousmaterial is subjected to said acid action at a, temperature of about18-25 C., in which said acid treatment is substantially maintained atsaid temperature of about 18-25" C. in which said acid treatedkeratinous material is subjected to the action of said aqueous solutioncontaining said NazS and said NazSzO; at a temperature of about 20-60(3., and in which 10 a. said NmS-NazSzO; reaction mass" is substantiallymaintained at said last mentioned temperature.

10. Improvement in accordance with claim 9 "pin which the combined Na2S-Na2SzO4 concentration in said aqueous medium is from about 0:2 to 1%.11. Improvement in accordance with claim 10 "in which said keratinousmaterial is subjected to the action of one member of the ;:groupconsisting of about 13 N HsPO4 and about 8 N -H2SO4, in which said acidtreated keratinous material is subjected to the action of said aqueousNazS--Na2Sz04 solution at a pH oi' about 9.5, in which the said lastmentioned pH is substan- 5 tially maintained in the NmS-NazSzO; reactionmass until the keratinous materialiis substantially dissolved, and inwhich the combined Na2S-Na2S2O4 concentration in said medium issufficient for cystine reduction.

ERNEST C. KOERNER.

REFERENCES cITEn The following references are of "record in the file ofthis patent:

UNITED STATES PATENTS

1. IN THE RECOVERY OF PROTEIN FROM KERATINOUS MATERIALS THE IMPROVEMENTWHICH COMPRISES SUBJECTING A KERATINOUS MATERIAL, ACID PRE-TREATED FORNA2S CYSTINE REDUCTION, TO THE ACTION OF AN AQUEOUS SOLUTION, CONTAININGPER DRY WEIGHT OF KERATINOUS MATERIAL PRESENT ABOUT 3-5% NA2S AND ABOUT3-6% NA2S204 AT A PH OF ABOUT 9 TO 10 AND AT A TEMPERATURE OF LESS THAN60*C., THE AMOUNT OF AQUEOUS MEDIUM BEING SUFFICIENT TO SUBSTANTIALLYCOVER THE KERATINOUS MATERIAL PRESENT AND THE COMBINES NA2S-NA2S204CONCENTRATION IN SAID MEDIUM BEING SUFFICIENT FOR CYSTINE REDUCTION OFSAID KERATINOUS MATERIAL, MAINTAINING THE REACTION MASS AT SAID PH ANDAT SAID TEMPERATURE UNTIL THE KERATINOUS MATERIAL IS SUBSTANTIALLYDISSOLVED AND RECOVERING PROTEIN FROM SAID SOLUTION.